Opiates are important for the treatment of pain, but their abuse presents a major health problem due to their association with the recent HIV epidemic. Understanding the actions opiates in vivo is crucial to their rational use clinically and the development of potential treatments for abuse. Opiates and the opioid peptides act through a family of receptors, of which at least three opioid receptors have been cloned: mu, delta and kappa1. Studies on the actions of these receptors carried out in tissue culture have provided important information regarding their biochemistry and second messenger systems. However, this approach cannot address many questions regarding their actions within an integrated nervous system. The current application proposes to explore the actions of these receptors in vivo. Antisense approaches will be utilized to correlate the cloned opioid receptors with opioid pharmacology. Initial studies from our laboratory demonstrate that antisense oligodeoxynucleotides (ODN) directed against delta, mu or kappa1 receptors selectively and specifically downregulate their own receptor without interfering the function of the others. Mismatch ODN controls are inactive. This approach will be validated, looking at the uptake and stability of the various ODN's in tissue culture and in vivo as well as establishing the criteria to optimize their use. Antisense ODN's then will be used to examine the receptors responsible for a variety of opioid actions, including analgesia, respiratory depression and the inhibition of gastrointestinal transit. The second component of this application addresses the issue of tolerance. This work will expand on previous observations that NMDA antagonists and nitric oxide synthase (NOS) inhibitors can prevent or reverse morphine tolerance. Additional agents will be examined and the role of NOS studied using a knockout mouse. Finally, regional interactions important in opioid analgesia will be explored. Opiates act through the activation of multiple brain and spinal regions simultaneously. These interactions and the existence of synergy will be explored. Studies demonstrating intrinsic brainstem synergy will be expanded and additional experiments will explore the mu/delta synergy between brainstem regions recently observed by our group. Overall, this proposal will provide important information correlating recent advances in the molecular biology of opioid receptors with their pharmacology.